Sterilization indicator

ABSTRACT

The disclosed technology relates to a sterilization indicator and a process to concentrate signal generated by constraining it to a minimal surface, in a minimal volume and minimal pH and growth buffering or mediating influences. The sterilization indicator may comprise a carrier  12 , the carrier having a first surface  14  and a second surface  16 ; a support  20 , the support having a first section  22  and a second section  24 , the carrier  12  overlying the first section  12  of the support  20 , the second surface  16  of the carrier being adhered to the first section  22  of the support  20 ; and a biological indicator  30  supported by the carrier  12 . The second section  24  of the support  20  may be of sufficient dimension to permit handling the sterilization indicator  10  without contacting the biological indicator  30 . A process for making the sterilization indicator is disclosed. Processes for using the sterilization indicator are disclosed.

This application is a divisional of U.S. patent application Ser. No.11/533,487 filed on Sep. 20, 2006, which is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The disclosed technology relates to a sterilization indicator, and to aprocess for making the sterilization indicator. The disclosed technologyrelates to a sterilization process using the sterilization indicator todetermine the effectiveness of sterilization.

BACKGROUND

Primarily in the health care industry, but also in many other commercialand industrial applications, it is often necessary to monitor theeffectiveness of the processes used to sterilize equipment such asmedical and non-medical devices, instruments and other articles andmaterials. It is often standard practice in these sterilizationprocesses to include a sterilization indicator in the batch of articlesto be sterilized. This allows a direct approach to assay the lethalityof the sterilization process.

Conventional sterilization indicators typically contain a biologicalindicator. The biological indicator may comprise one or more testorganisms which are designed to be more resistant to the sterilizationprocess than the organisms to be destroyed by the sterilization. Thesetest organisms are usually bacterial spores. Conventional sterilizationindicators may be available in two forms.

The first of these forms involves the use of a substrate whereinbacterial spores are directly applied or inoculated on the substrate.The substrate may be fully covered with the spores. Any physicalhandling by the user may result in spores being lost from the substrate,transferred to the user, or potentially contaminating or beingcontaminated by the surrounding area. It has been proposed to providespecial clips to allow the user to handle the substrate. However, theseclips often hinder the sterilization process and may result in a faultytest result. Also, minimum size constraints for these substratestypically lead to the requirement for relatively large volumes ofincubation medium, for example, from about 5 to about 10 milliliters(ml), and relatively long incubation periods, for example, from about 2to about 7 days.

The second of these forms involves a self-contained sterilizationindicator. These sterilization indicators typically contain thebacterial spores and the incubation medium in a single container, but inseparate compartments. The spores are subjected to the sterilizationprocess. Following sterilization, the container is activated so that anysurviving spores may come into contact with the incubation medium todetermine the effectiveness of the sterilization. These sterilizationindicators may be useful in gaseous sterilization processes, but aretypically not suitable for liquid sterilization processes.

A major drawback with each of these sterilization indicators relates tothe time delay in obtaining results for the sterilization test. Thesesterilization indicators normally require that the bacterial spores becultured for at least two and often up to about seven days to assureadequate detection of any surviving spores. During this time, thearticles that went through the sterilization process and are underevaluation should not be used until the results of the spore viabilitytest have been determined. However, many health care facilities havelimited resources and must reuse their “sterilized” instruments within24-48 hours and often immediately. In such settings, the two to sevenday holding period for sterility verification may be impractical, costlyand inefficient.

Thus, a problem that has been presented by the art is to provide asterilization indicator that minimizes or eliminates the handling of thebiological indicator and accurately detects the effectiveness of asterilization process within a relatively short period of time. It wouldbe advantageous if this sterilization indicator could be adaptable toliquid sterilization processes as well as gaseous sterilizationprocesses. The disclosed technology, in at least one embodiment, mayprovide a solution to this problem.

SUMMARY

The disclosed technology may relate to a sterilization indicator,comprising: a carrier, the carrier having a first surface and a secondsurface; a support, the support having a first section and a secondsection, the carrier overlying the first section of the support, thesecond surface of the carrier being adhered to the first section of thesupport; and a biological indicator supported by the carrier, the secondsection of the support being of sufficient dimension to permit handlingthe sterilization indicator without contacting the biological indicator.

The disclosed technology may relate to a sterilization indicator kit,comprising: a first compartment, containing the above-indicatedsterilization indicator, the first compartment being adapted to permitthe sterilization indicator to be brought into contact with asterilization medium during sterilization; and a second compartmentcontaining an incubation medium, the second compartment being adapted tomaintain the incubation medium separate from the sterilization indicatorduring sterilization, and the second compartment being adapted to permitthe incubation medium to contact the sterilization indicator after thesterilization indicator has been exposed to the sterilization medium.

The disclosed technology may relate to a process for making theabove-indicated sterilization indicator, comprising: applying thebiological indicator to the carrier; and adhering the carrier to thesupport.

The disclosed technology may relate to a sterilization process,comprising: exposing an article to be sterilized and the above-indicatedsterilization indicator to a sterilization medium.

The disclosed technology may relate to a process for determining theeffectiveness of sterilization, comprising: exposing an article to besterilized and the above-indicated sterilization indicator to asterilization medium, the biological indicator comprising at least onetest organism; and contacting the carrier with an incubation mediumafter sterilization to determine whether the sterilization is effective.

The disclosed technology may relate to a process for determining theeffectiveness of sterilization, comprising: exposing at least onearticle to be sterilized and the above-indicated sterilization indicatorto a sterilization medium, the biological indicator comprising at leastone enzyme; and contacting the carrier with at least one enzymesubstrate to determine whether the sterilization is effective.

BRIEF DESCRIPTION OF THE DRAWINGS

In the annexed drawings, like parts and features have like references.

FIG. 1 is a plan view of a schematic illustration of one embodiment ofthe disclosed sterilization indicator.

FIG. 2 is a schematic illustration of a side elevation of thesterilization indicator depicted in FIG. 1.

FIG. 3 is an exploded schematic illustration of an apparatus fordetermining the effectiveness of sterilization, the apparatus containingtwo compartments, the foregoing sterilization indicator being positionedin one compartment and an incubation medium being positioned in theother compartment.

FIG. 4 is a plot of recoverable spores for samples of the disclosedsterilization indicator in Example 1, the plot indicating no significantdifference in the number of viable spores following sonication ascompared to prior to sonication.

FIG. 5 is a plot of pH as a function of growth of spores of Geobacillusstearothermophilus exposed to incubation media of different volumes andbuffering capacities as disclosed in Example 2.

DETAILED DESCRIPTION

The term “sterilization” may refer to rendering a substance incapable ofreproduction, metabolism and/or growth. While this is often taken tomean total absence of living organisms, the term may be used herein torefer to a substance free from living organisms to a degree previouslyagreed to be acceptable. Unless otherwise indicated, the termsterilization may be used herein to also refer to methods and proceduresless rigorous than sterilization, for example, disinfection,sanitization, and the like. The sterilization indicator and theprocesses and apparatus described herein may be used in health carefields, scientific fields, and the like. These may be used in commercialand industrial applications where sterilization, disinfection,sanitization, and the like, may be desired, for example, foodprocessing, pharmaceutical manufacturing, and the like.

The sterilization process for which the disclosed sterilizationindicator may be used may be any sterilization process. These mayinclude sterilization processes wherein the sterilization medium orsterilant may be steam, dry heat, radiation, as well as one or moregaseous sterilants, one or more liquid sterilants, and the like. Theradiation may comprise electron beam or any electromagnetic spectraincluding ionizing radiation, pulsed white or ultraviolet light,microwave, and the like. The radiation may comprise gamma or betaradiation. The gaseous sterilants may comprise ethylene oxide, gaseoushydrogen peroxide, and the like. The liquid sterilants may compriseformalin (formaldehyde gas dissolved in water and optionally containingmethanol to inhibit the formation of toxic substances), glutaraldehyde,peracetic acid, liquid hydrogen peroxide, and the like.

The sterilization indicator may be used to examine the lethality ofsterilants against any microorganism with less resistance to thesterilization process than the biological indicator used with thesterilization indicator. These microorganisms may include bacteria suchas Escherichia coli, Legionella sp., Campylobacter sp., and otherenteric bacteria, as well as Staphylococcus and Streptococcus speciesand other human pathogenic microorganisms such as Cryptosporidium.

The sterilization indicator may be described with reference to FIGS. 1and 2. Referring to these figures, sterilization indicator 10 maycomprise carrier 12, the carrier 12 having a first surface 14 and asecond surface 16; support 20, the support 20 having a first section 22and a second section 24, the carrier 12 overlying the first section 22of the support 20, the second surface 16 of the carrier 12 being adheredto the first section 22 of the support 20; and biological indicator 30supported by the carrier 12. The biological indicator 30 may besupported by or adhered to the first surface 14 of the carrier 12. Thesecond section 24 of the support 20 may be of sufficient dimension topermit handling the sterilization indicator 10 without contacting thebiological indicator 30. That is, the second section 24 may be ofsufficient dimension to function as a handle thereby permittingfacilitated aseptic handling of the sterilization indicator 10.

The carrier 12 may be in the form of a relatively flat substrate whichis depicted in the drawings as being in the form of a circle. However,it is to be understood that the carrier 12 may have any desired shape orform, for example, square, rectangle, oval, and the like. The carrier 12may have a prismatic cross-section. The carrier 12 may have a thicknessthat is relatively small, for example, from about 0.001 to about 3 mm,and in one embodiment from about 0.01 to about 2 mm, and in oneembodiment from about 0.05 to about 1.5 mm, and in one embodiment fromabout 0.1 to about 1 mm. The area of the first surface 14 of the carrier12, which provides support for the biological indicator 30, may berelatively small, for example, the area may be in the range from about 1to about 80 mm², and in one embodiment from about 2 to about 70 mm², andin one embodiment from about 3 to about 60 mm², and in one embodimentfrom about 5 to about 50 mm². An advantage of such a small area is thatthe size of the biological indicator 30 may be relatively small, andconsequently the amount of incubation medium needed to incubate thebiological indicator may be relatively small and the time requirementfor incubation may be relatively short.

The carrier 12 may comprise a porous material or a non-porous material.The carrier may comprise a solid carrier. The carrier may comprise anymaterial that does not dissolve or deteriorate during the sterilizationor incubation processes. The carrier 12 may comprise paper, metal,glass, ceramics, plastic, membranes, or a combination of two or morethereof. The metal may comprise aluminum or steel. The plastic maycomprise a polyolefin, polystyrene, polycarbonate, polymethacrylate,polyacrylamide, polyimide, polyester, and the like. The carrier 12 maycomprise a film. The carrier may be in the form of a spun or unwovenfelt. The carrier may comprise a mat of compressed fibers. The carriermay comprise a porous material made of sintered glass, glass fibers,ceramic, synthetic polymer, or a combination of two or more thereof. Thecarrier may comprise filter paper or absorbent paper. The carrier maycomprise a cellulose pad.

The support 20 may comprise any material that does not dissolve ordisintegrate during the sterilization or incubation processes. Thesupport may comprise metal, glass, ceramic, plastic, or a combinationthereof. The support may comprise aluminum or stainless steel. Thesupport may comprise polystyrene, polyolefin (e.g., polypropylene,polyethylene), and the like. The support 20 may be flexible or rigid.The support 20 may be foldable. The support 20 depicted in the drawingsis rectangular in shape, however, it is to be understood that thesupport may have any desired shape or form, for example, square, circle,oval, and the like. The length of the support 20 may be in the rangefrom about 0.2 to about 12 cm, and in one embodiment from about 0.2 toabout 10 cm, and in one embodiment from about 0.5 to about 7 cm, and inone embodiment from about 1 to about 5 cm, and in one embodiment fromabout 1.5 to about 3.5 cm. The width of the support 20 may be in therange from about 0.2 to about 2 cm, and in one embodiment from about 0.2to about 1.5 cm, and in one embodiment from about 0.25 to about 1 cm.The thickness of the support 20 may be in the range from about 0.02 toabout 3 mm, and in one embodiment from about 0.1 to about 2 mm. Thelength of the second section 24 may be in the range from about 0.2 toabout 12 cm, and in one embodiment from about 0.3 to about 11 cm, and inone embodiment from about 0.5 to about 10 cm, and in one embodiment fromabout 1 to about 7 cm, and in one embodiment from about 1.5 to about 4.5cm.

The support 20 may be in the form of a rectangular sheet or strip, thefirst section 22 of the support 20 comprising a minor part of the lengthof the carrier 20, the second section 24 of the support 20 comprising amajor part of the length of the support 20. The ratio of the length ofthe second section 24 to the length of the first section 22 may be inthe range from about 2:1 to about 12:1, and in one embodiment from about4:1 to about 8:1, and in one embodiment from about 5.5:1 to about 6.5:1.

The carrier 12 may be attached to the support 20 using sonic welding,heat sealing, an adhesive, or lamination. The carrier 12 may be attachedto the support 20 prior to or subsequent to applying the biologicalindicator 30 to the carrier 12. The carrier 12 may be attached to thesupport 20 subsequent to applying the biological indicator 30 to thesupport 12 using sonic welding or an adhesive. Sonic welding may involvefrictional binding of the support to the carrier. The adhesive may beany adhesive that is compatible with the carrier 12 and the support 20,and does not dissolve or deteriorate during the sterilization orincubation processes. The adhesive should not be lethal or inhibitory tothe organisms of interest. The adhesive may be a pressure sensitiveadhesive.

The sterilization indicator 10 may be used in any process wherein thebiological indicator 30 is exposed to a sterilization medium during asterilization process and then to an incubation medium to determinewhether the sterilization process was effective. The sterilizationindicator 10 may be used with any sterilization process, for example,sterilization processes employing gaseous or liquid sterilants. Thesterilization indicator 10 along with the articles to be sterilized maybe exposed to a sterilization medium during a sterilization process.Upon completion of the sterilization process, the sterilizationindicator 10 may be placed in a vial containing an incubation medium.The biological indicator 30 may then be incubated for a desired periodof time and then examined to determine whether the sterilization processwas effective.

The sterilization indicator 10 may be used in a self-containedsterilization indicator kit comprising a container with two separatecompartments. One of the compartments may contain the sterilizationindicator 10. The other compartment may contain an incubation medium. Inuse, the kit and the articles to be sterilized may be exposed to thesterilization medium. Then following sterilization, the kit may beactivated so that the biological indicator 30 comes into contact withthe incubation medium sufficiently to determine whether thesterilization process was effective. These kits may be used with anysterilization process wherein the biological indicator may be exposed tothe sterilization medium, for example, sterilization processes employinggaseous sterilants.

The self-contained sterilization indicator kit may be in the formdepicted in FIG. 3. Referring to FIG. 3, the kit 40 comprises taperedtube 42, inner compartment 44, and closure cap 46. The closure cap 46includes projections 48. An annular space 43 is formed between the innersurface of tapered tube 42 and the outer surface of the innercompartment 44, the annular space 43 forming another interiorcompartment. The sterilization indicator 10 is positioned in the annularspace 43. The incubation medium is contained in the inner compartment44. The tapered tube 42 and the closure cap 46 may be made from anymaterial that is compatible with the conditions and chemistries used inthe sterilization process. These materials may include polycarbonate,polyolefins, polyamide, polymethacrylates, polymethylpentenes,polyesters, and the like. The inner compartment 44 may be in the form ofa glass or frangible glass ampoule. Further details on the constructionof tapered tube 42, inner compartment 44 and closure cap 46 may be foundin U.S. Pat. No. 4,304,869, which is incorporated herein by reference.During sterilization, the kit 40, along with the articles to besterilized, is exposed to the sterilization medium. When sterilizationhas been completed, the closure cap 46 is pressed downwardly into thetapered tube 42. The projections 48 press against the inner compartment44 and cause it to rupture. This allows the incubation medium to contactbiological indicator 30. After incubation for a predetermined time, thesterilization indicator 10 may be removed and the extent ofsterilization may be determined by detecting change in the biologicalindicator 30.

The biological indicator 30 may comprise one or more test organisms.Alternatively, the biological indicator 30 may comprise one or moreenzymes. The one or more enzymes may be derived from and/or isolatedfrom one or more test organisms. The biological indicator 30 maycomprise one or more test organisms in combination with one or moreenzymes.

The test organism may comprise any organism whose resistance to theintended sterilization process exceeds that of the other organisms thatare to be destroyed by the sterilization process. The type of testorganism used as the biological indicator 30 may be dependent upon avariety of factors exemplified by, but not limited to, the type ofsterilization process being used. The test organism may be amicroorganism. The strains that may be used may be those that are themost resistant to the process used for sterilization. The testmicroorganism may comprise bacteria. The bacterial microorganisms may bethose which form endospores, i.e., bacterial spores. The test organismmay comprise bacteria of the Bacillus or Clostridia genera. These mayinclude Geobacillus stearothermophilus, Bacillus atrophaeus, Bacillussubtilis, Bacillus pumilus, Bacillus coagulans, Clostridium sporogenes,Bacillus subtilis globigii, Bacillus cereus, Bacillus circulans, and thelike. The bacteria may comprise fungi, mycobacteria, protozoa,vegetative bacteria, and the like. Examples of fungi that may be usedmay include Aspergillus niger, Candida albicans, Trichophytonmentagrophytes, Wangiella dermatitis, and the like. Examples ofmycobacteria that may be used may include Mycobacterium chelonae,Mycobacterium gordonae, Mycobacterium smegmantis, Mycobacterium terrae,and the like. Examples of protozoa that may be used may include Giardialamblia, Cryptosporidium parvum, and the like. Examples of vegetativebacteria that may be used may include Aeromonas hydrophila, Enterococcusfaecalis, Streptococcus faecalis, Enterococcus faecium, Streptococcuspyrogenes, Escherichia coli, Klebsiella (pneumoniae), Legionellapneumophila, Methylobacterium, Pseudomonas aeruginosa, Salmonellacholeraesuis, Helicobacter pylori, Staphylococcus aureus, Staphylococcusepidermidis, Stenotrophomonas maltophilia, and the like. Organisms suchas Geobacillus stearothermophilus, Bacillus atrophaeus, Bacillussubtilis, Bacillus coagulans, Clostridium sporogenes, and the like, maybe used for determining the efficacy of moist heat sterilization(autoclaving), with Geobacillus stearothermophilus being especiallyuseful.

Microorganisms such as vegetative bacteria, vegetative cells and/ortheir constituent parts, which may be used as the test organism, may bedeposited on the carrier 12 and survive drying and storage whendeposited in the presence of one or more excipients. Excipients may bedefined as a broad class of generally inert compounds that may be usedto stabilize labile entities. A subclass of excipients that may be usedincludes the carbohydrates, for example, oligomeric and polymericsaccharides. An example of such a compound may be trehalose which is adisaccharide. High concentrations of trehalose in the tissues of certainorganisms may allow the organisms to survive in a state of waterdeficiency. Trehalose may be used to revive functional cellularcomponents after dehydration. Trehalose may provide stability tomembranes and other macromolecular structures essential to the viabilityof a cell under extreme environmental conditions (e.g., freeze drying).Other stabilizing excipient compounds may include simple sugars (e.g.sucrose, glucose, maltose, and the like) and long chain polymers (e.g.dextrans, starch, agarose, cellulose, and the like). Othernon-carbohydrate based excipients may include proteins, phosphonates,buffering agents, waxes, lipids, oils as well as other hydrocarbon basedmaterials.

In addition to the simulative organisms selected on the basis of theiracceptance as representing the most resistant organism (e.g. Geobacillusstearothermophilus), the biological indicator 30 may further comprisenon self-replicating agents and/or sub-cellular components or productsof cells. These may be used because of their clinical significance orbecause of their use as agents of bioterrorism. These organisms maycomprise strains which may now have resistance to normal means ofantibiotic treatment or chemical disinfection due to natural or man-mademodifications. Examples of the former type may include VREs (VancomycinResistant enterococci), MSRAs (Methicillin Resistant Staphylococcusaureus), Mycobacterium cheloni, and the like. These may be used becausethe VREs and MRSAs have developed resistance to therapeuticcountermeasures (e.g., antibiotic resistance) and M. cheloni hasdeveloped resistance to some modes of disinfection (e.g., glutaraldehyderesistance).

The biological indicator 30 may comprise one or more emerging organismsfor which there may not yet be a simulative alternative. These mayrepresent a special risk or challenge to therapeutic course of action ordisinfection. Examples of these organisms may include prions. Prions arenot living organisms, per se, but their function as disease causingagents may be related to their structure and this structure/functionrelationship may be employed to determine their relative infectivity.Other non-autonomous agents (e.g. viruses) as well as sub cellularelements and proteinaceous prions may be used as the biologicalindicator 30.

The carrier 12 may be inoculated with the test organism by preparing anaqueous suspension or dispersion comprising the test organism. Theaqueous suspension or dispersion may comprise, for example, bacterialspores at a concentration ranging, for example, from about 10⁵ to about10¹⁰ colony forming units (cfu) per milliliter, and in one embodimentfrom about 10⁷ to about 10⁹ cfu per milliliter. An aliquot of thesuspension or dispersion may be placed on the carrier 12. For example, asuspension or dispersion of B. subtilis spores in water may be preparedto yield a desired number of spores per aliquot for inoculating thecarrier 12. The spores may be allowed to dry on the carrier. An air flowmay be used to dry the spores on the support, such as, for example, byplacing the carrier in a laminar flow-hood to hasten the drying process.The method of drying the spores on the carrier may include allowing thespores to air dry by leaving them stand, placing the spores in adesiccator containing a desiccant such as calcium chloride, placing thespores in a laminar-flow hood, and the like. The number of colonyforming units supported by the carrier 12 may be in the range from about10⁴ to about 10⁷ cfu per square millimeter of support (cfu/mm²), and inone embodiment in the range from about 10⁵ to about 10⁶ cfu/mm².

The sterilization indicator 10 may be used by subjecting it to the samesterilization medium and treatment as the articles for which sterileconditions may be sought. Heat may be applied and/or a gas, liquid,steam, or chemical and/or physical agent may pass into the area wherethe biological indicator 30 is located thereby exposing the biologicalindicator 30 to the same sterilization process or agent as the articlesbeing sterilized. Following sterilization, an incubation medium may bebrought into contact with the biological indicator 30. The incubationmedium may be referred to as a growth medium. The incubation medium maybe in the form of a solid or a liquid. The incubation medium maycomprise a buffered aqueous solution although an advantage of thedisclosed technology is that the buffer capacity of the incubationmedium may be reduced so that the biological indicator may be moresensitive to pH shifts, redox potentials, enzymatic activity, and thelike. Any procedure whereby the biological indicator is brought intocontact with the incubation medium under conditions which allow forgrowth of the test organism, if it still exists, may be used. Theincubation medium may be present in the sterilization chamber in powderor tablet form and, after sterilization, sterile water may be added suchthat the biological indicator comes into contact with the aqueousincubation medium.

The incubation medium may comprise one or more nutrient sources. Thenutrient source may be used to provide energy for the growth of any ofthe test organisms that may survive the sterilization process. Examplesof the nutrient sources may include pancreatic digest of casein,enzymatic digest of soybean meal, sucrose, dextrose, yeast extract,L-cystine, and mixtures of two or more thereof. A microbial growthindicator, which changes color or native state, in the presence ofviable test organisms may be used with the incubation medium. The growthindicator may be dispersed or solubilized in the incubation medium andimpart an initial color to the incubation medium. The growth indicatormay also impart a color change in the incubation medium uponmicroorganism growth. Growth indicators which may be employed includepH-sensitive dye indicators (such as bromothymol blue, bromocresolpurple, phenol red, etc. or combinations thereof), oxidation-reductiondye indicators (such as methylene blue, etc.), enzyme substrates, ormixtures of two or more thereof. The enzyme substrate may comprise anyenzyme substrate whose activity correlates with one or more enzymes thatmay be present in the test organism. The enzyme substrates that may beused may include those discussed below. The use of these microbialgrowth indicators may result in a change in color in response to aphenomenon of microorganism growth, such as changes in pH,oxidation-reduction potentials, enzymatic activity, as well as otherindications of growth. The incubation medium may further comprise one ormore pH buffers, one or more neutralizers, one or more agents formaintaining osmotic equilibrium, or a mixture of two or more thereof.The pH buffers may include K₂HPO₄, KH₂PO₄, (NH₄)₂HPO₄,2,2-Bis(hydroxylmethyl)-2,2′,2″-nitrilothiethanol (Bis Tris), 1,3-Bis[tris(hydroxymethyl)methylamino]propane (Bis-Tris Propane),4-(2-Hydroxyethyl)piperazine-ethanesulfonic acid (HEPES),2-Amino-2-(hydroxymethyl)-1,3-propanediol (Trizma, Tris base),N-[Tris(hydroxymethyl)methyl]glycine (Tricine), Diglycine (Gly-Gly),N,N-Bis(2-hydroxyethyl)glycine (Bicine), N-(2-Acetamido)iminodiaceticacid (ADA), N-(2-Acetamido)-2-aminoethanesulfonic acid (aces),1,4-Piperazinediethanesulfonic acid (PIPES),β-Hydroxy-4-morpholinepropanesulfonic acid (MOPSO),N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-(N-Morpholino)propanesulfonic acid (MOPS),2-[(2-Hydroxy-1,1-bis(hydroxylmethyl)ethyl)amino]ethanesulfonic acid(TES), 3-(N,N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid(DIPSO), 4-(N-Morpholino)butanesulfonic acid (MOBS),2-Hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid(TAPSO), 4-(2-Hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acidhydrate (HEPPSO), Piperazine-1,4-bis(2-hydroxypropanesulfonic acid)dihydrate (POPSO), 4-(2-Hydroxyethyl)-1-piperazine propanesulfonic acid(EPPS), N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid) (HEPBS),[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid(TAPS), 2-Amino-2-methyl-1,3-propanediol (AMPD),N-tris(Hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), 2-(Cyclohexylamino)ethanesulfonic acid (CHES),3-(Cyclohexylamino)-2-hydroxyl-1-propanesulfonic acid (CAPSO),2-Amino-2-methyl-1-propanol (AMP), 3-(Cyclohexylamino)-1-propanesulfonicacid (CAPS), 4-(Cyclohexylamino)-1-butanesulfonic acid (CABS),2-(N-Morpholino)ethanesulfonic acid hydrate (MES),N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), and mixtures of twoor more thereof. The neutralizers may include but are not limited tosodium thioglycollate, sodium thiosulfate, catalase, sodium bisulfate,sodium bisulfite lecithin, polysorbate 20, polysorbate 80, calciumbicarbonate, and mixtures of two or more thereof. The agents formaintaining osmotic equilibrium may include sodium salt, potassiumsalts, magnesium salts, manganese salts, calcium salts, metallic salts,sodium chloride, potassium chloride, magnesium sulfate, iron chloride,and mixtures of two or more thereof. The incubation medium may comprisean aqueous composition comprising: water; from about 0.01 to about 100grams per liter of water (g/l), and in one embodiment from about 0.1 toabout 50 g/l, of one or more nutrient sources; from about 1.0×10⁻⁵ toabout 10 g/l, and in one embodiment from about 1.0×10⁻⁴ to about 1.0 g/lof one or more microbial growth indicators; up to about 5000 g/l, and inone embodiment from about 0.001 to about 5000 g/l, and in one embodimentfrom about 0.1 to about 1000 g/l, of one or more pH buffers; up to about100 g/l, and in one embodiment from about 0.01 to about 100 g/l, and inone embodiment from about 0.1 to about 50 g/l, of one or moreneutralizers; up to about 50 g/l, and in one embodiment from about 0.1to about 50 g/l, and in one embodiment from about 0.1 to about 25 g/l,of one or more agents for maintaining osmotic equilibrium.

The incubation medium may have a relatively low buffering capacity inthe range from about −0.001 to about −0.070 mol H⁺, and in oneembodiment in the range from about −0.01 to about −0.070 mol H⁺, and inone embodiment in the range from about −0.01 to about −0.014 mol H⁺.This buffering capacity may be provided by formulating the incubationmedium with the indicated low buffering capacity or by diluting apre-formulated incubation medium having a higher buffering capacity withwater or other liquid. In the prior art, incubation media have typicallybeen formulated to enhance the growth of organisms. This may cause theorganisms to expend more energy on growth and division and less energyon pumping out accumulating acidic waste and other by-products producedby metabolism. The buffers within an incubation medium may be used tokeep the medium sufficiently neutral or alkaline to facilitate thetransport of acidic by-products (protons) across the cell membrane. Theincubation medium used with the disclosed sterilization indicator mayfocus on how rapidly growth may be detected. By employing a reducedbuffering capacity in the range from about −0.001 to about −0.010 molH⁺, and in one embodiment in the range from about −0.005 to about −0.007mol H⁺, the incubation medium may be more sensitive to small shifts inpH, redox potentials and/or enzymatic activity.

The incubation medium may comprise a nutrient broth, D/E neutralizingbroth, Davis minimal medium, sterility test broth, as well as anysoybean-casein digest or beef extract based media. These may include anaqueous solution of soybean-casein digest broth, fluid thioglycollateand Dextrose Tryptone (Difco Laboratories, Inc.). A modified tryptic soybroth base, without glucose, may be used. If enzymatic activity is beingmeasured, the incubation medium may comprise water, an enzyme substrate,and optionally pH buffers.

An example of an incubation medium that may be used is Bacto™ TrypticSoy Broth which contains pancreatic digest of casein (17.0 g/l),enzymatic digest of soybean meal (3.0 g/l), sodium chloride (5.0 g/l),dipotassium phosphate (2.5 g/l), and dextrose (2.5 g/l). Theseingredients may be dispersed or dissolved in water. The concentrationsexpressed in terms of g/l refer to grams of ingredient per liter ofwater. Pancreatic digest of casein, enzymatic digest of soybean meal,and dextrose provide energy sources for growth of the microorganism.These may be referred to as nutrient sources. Sodium chloride may beused to maintain an osmotic equilibrium in the liquid medium.Dipotassium phosphate may act as a pH buffer. Phenol red, for example,which is a pH-sensitive dye, may be added (18 mg/l) to the Bacto™Tryptic Soy Broth formulation. The dye may be useful as an indicator ofpH shift in the incubation medium resulting from microorganism growth.

Another example of an incubation medium that may be used is BBL™ FluidThioglycollate Medium. This incubation medium contains pancreatic digestof casein (15.0 g/l), yeast extract (5.0 g/l), dextrose (5.5 g/l),sodium chloride (2.5 g/l), L-cystine (0.5 g/l), sodium thioglycollate(0.5 g/l), and resazurin (1.0 mg/l). These ingredients may be dispersedor dissolved in water. The pancreatic digest of casein, yeast extract,dextrose, and L-cystine are nutrient sources which may provide energyfor microorganism growth. The sodium chloride may be used to maintainosmotic equilibrium in the incubation medium. Sodium thioglycollate maybe used as a neutralizer. Resazurin may be used as anoxidation/reduction dye indicator. Other nutrient sources, osmoticmediators and general ingredients known to those practiced in the artmay be substituted for listed ingredients.

An advantage of the disclosed technology is that the size of thebiological indicator 30 may be relatively small and thus the volume ofincubation medium needed to incubate any surviving test organisms on thecarrier 12 may be relatively small. This may result in an incubationperiod that may be relatively short. Thus, in one embodiment, thebiological indicator 30 may be supported by a carrier 12 that isrelatively small, the carrier 12 having a surface area in the range fromabout 1 to about 80 mm², and in one embodiment from about 5 to about 50mm². The number of spores supported by the carrier 12, beforesterilization, may be in the range from about 10⁴ to about 10⁷ cfu/mm²,and in one embodiment in the range from about 10⁵ to about 10⁶ cfu/mm².The volume of incubation medium needed to incubate the biologicalindicator 30 after sterilization may be in the range from about 0.1 toabout 5 ml, and in one embodiment from about 0.1 to about 4 ml, and inone embodiment from about 0.1 to about 3 ml, and in one embodiment fromabout 0.1 to about 2 ml, and in one embodiment from about 0.1 to about1.5 ml, and in one embodiment from about 0.3 to about 1 ml. The timerequired to incubate the biological indicator 30 after sterilization maybe in the range from about 0.1 to about 48 hours, and in one embodimentfrom about 0.1 to about 36 hours, and in one embodiment from about 0.1to about 24 hours, and in one embodiment from about 0.1 to about 18hours, and in one embodiment from about 0.1 to about 15 hours, and inone embodiment from about 0.1 to about 12 hours, and in one embodimentfrom about 0.1 to about 10 hours, and in one embodiment from about 0.1to about 8 hours, and in one embodiment from about 0.1 to about 6 hours,and in one embodiment from about 0.1 to about 5 hours, and in oneembodiment from about 0.1 to about 4 hours.

The enzyme that may be used as the biological indicator 30 may compriseany enzyme, including extracellular or intracellular enzymes, whoseactivity may correlate with the viability of at least one test organism.By “correlate” it is meant that the enzyme activity, over background,may be used to predict future growth of a test organism. The enzyme maybe one which following a sterilization cycle, which is sublethal to thetest organism, remains sufficiently active to react with an enzymesubstrate within a desired period of time, for example, from about 4 toabout 164 hours, and in one embodiment from about 4 to about 84 hours,yet be inactivated or appreciably reduced in activity following asterilization which would be lethal to the test organism.

The following test may be useful in identifying those enzymes having therequisite characteristics to be useful as the biological indicator 30.The enzyme when subjected to sterilization conditions which would bejust sufficient to decrease the population of about 1×10⁶ test organismsby about 6 logs (i.e., to a population of about zero as measured by lackof outgrowth of the test organisms), may have residual enzyme activitywhich is equal to “background” as measured by reaction with an enzymesubstrate; however, the enzyme upon being subjected to sterilizationconditions sufficient only to decrease the population of about 1×10⁶test organisms by at least about 1 log, but less than about 6 logs, mayhave enzyme activity greater than “background” as measured by reactionwith the enzyme substrate. The enzyme substrate may be a substance, ormixture of substances, which when acted upon by the enzyme produces adetectable, e.g., fluorescent or colored, enzyme-modified product. Theenzyme activity may be measured by the amount of detectableenzyme-modified product produced. The enzyme may be one which hassufficient activity, following sterilization conditions insufficient todecrease the population of the test organism by about 6 logs, to reactwith the enzyme substrate and produce a detectable amount ofenzyme-modified product within a period of time in the range from about0.1 to about 48 hours, and in one embodiment in the range from about 0.1to about 12 hours, and in one embodiment in the range from about 0.1 toabout 4 hours.

The activity of the biological indicator 30 after sterilizationconditions that are insufficient to decrease the microorganismpopulation by about 6 logs, may be at least about 2 percent greater thanbackground, and in one embodiment at least about 5 percent greater thanbackground, and in one embodiment at least about 10 percent greater thanbackground. The residual enzyme activity level which is defined as“background” may be higher than that achieved by the spontaneousconversion of enzyme substrate to product after the enzyme has beeninactivated.

Enzymes which may be used in the biological indicator 30 may include,but not limited to, hydrolytic enzymes from spore-formingmicroorganisms. These enzymes may include beta-D-glucosidase,alpha-D-glucosidase, alkaline phosphatase, acid phosphatase, butyrateesterase, caprylate esterase lipase, myristate lipase, leucineaminopeptidase, valine aminopeptidase, chymotrypsin, phosphohydrolase,alpha-D-galactosidase, beta-D-galactosidase,alpha-L-arabinofuranosidase, N-acetyl-beta-glucosaminidase,beta-D-cellobiosidase, alanine aminopeptidase, proline aminopeptidase,tyrosine aminopeptidase, phenylalanine aminopeptidase,beta-D-glucuronidase, and a fatty acid esterase, derived fromspore-forming microorganisms, such as Candida, Bacillus or Clostridiumspecies of microorganisms.

Enzymes from Geobacillus stearothermophilus that may be used may includealpha-D-glucosidase, beta-D-glucosidase, alkaline phosphatase, acidphosphatase, butyrate esterase, caprylate esterase lipase, leucineaminopeptidase, chymotrypsin, phosphophydrolase, alpha-D-galactosidase,beta-D-galactosidase, alanine aminopeptidase, tyrosine aminopeptidase,and phenylalanine aminopeptidase and a fatty acid esterase. Enzymes fromBacillus subtilis that may be used include alpha-L-arabinofuranosidase,beta-D-glucosidase, N-acetyl-beta-glucosaminidase,beta-D-cellobiosidase, alanine aminopeptidase, proline aminopeptidase,tyrosine aminopeptidase, leucine aminopeptidase and phenylalanineaminopeptidase.

Beta-D-glucosidase and alpha-L-arabinofuranosidase from Bacillussubtilis may be used in the monitoring of ethylene oxide sterilization.Alpha-D-glucosidase from Geobacillus stearothermophilus may be used tomonitor steam sterilization conditions.

An enzyme substrate may be a substance or mixture of substances whichwhen acted upon by an enzyme is converted into an enzyme-modifiedproduct. In general, the enzyme-modified product may be a luminescent,fluorescent, colored or radioactive material. However, the enzymesubstrate may comprise one or more compounds which when acted upon bythe enzyme, may yield a product which reacts with an additional compoundor composition to yield a luminescent, fluorescent, colored orradioactive material. When the enzyme substrate is to be included in thebiological indicator 30 during sterilization, the enzyme substrateshould not spontaneously break down or convert to a detectable productduring sterilization or incubation. For example, in sterilizationindicators used to monitor steam and dry heat sterilization, the enzymesubstrate should be stable at temperatures between about 20° C. andabout 180° C. Where the enzyme substrate is to be included with aconventional incubation medium, it should be stable in the incubationmedium, e.g., not autofluoresce in the incubation medium.

There are two basic types of enzyme substrates that may be used for thedetection of specific enzymes. The first type of enzyme substrate may beeither fluorogenic or chromogenic, and may be given a chemical formulasuch as, AB. When acted upon by the enzyme, AB, may break down to A+B.B, for example, may be either fluorescent or colored. A specific exampleof a fluorogenic substrate of this type may be 4-methylumbelliferylphosphate. In the presence of the enzyme phosphatase, the substrate maybe broken down into 4-methylumbelliferone and phosphate. Otherfluorogenic substrates of this type may include the derivatives of4-methylumbelliferyl, resorufin, and fluorescein. An example of achromogenic substrate of this type may be 5-bromo-4-chloro-3-indolylphosphate. In the presence of phosphatase, the substrate may be brokendown into indigo blue and phosphate. Other chromogenic substrates ofthis type may include derivatives of indoxyl, nitrophenol andphenolphthalein, where chromogenic indoxyl substrates may be broken downand a colorimetric response produced by the following chemical reaction,AB. When subsequently acted upon by the appropriate enzyme, AB, maybreak down to A+B. The color may then be obtained when BB occurs.

The second type of enzyme substrate may be given the chemical formulaCD, for example, which may be converted by a specific enzyme to C+D.However, neither C nor D may be fluorescent or colored, but D may becapable of being further reacted with compound Z to give a fluorescentor colored compound, thus indicating enzyme activity. A specificfluorogenic example of this type may be the amino acid lysine. In thepresence of the enzyme lysine decarboxylase, lysine may lose a moleculeof CO₂. The remaining part of the lysine may then be called cadaverine,which is strongly basic. A basic indicator such as 4-methylumbelliferonemay be incorporated and may fluoresce in the presence of a strong base.A chromogenic substrate of this type may be 2-naphthyl phosphate. Theenzyme phosphatase, may react with the enzyme substrate to yieldbeta-naphthol. The liberated beta-naphthol may react with a chromogenicreagent containing 1-diazo-4-benzoylamino-2,5-diethoxybenzene to producea violet color.

The enzyme substrate may be a fluorogenic compound, defined herein as acompound capable of being enzymatically modified, e.g., by hydrolysis,to provide a derivative fluorophor which has an appreciably modified orincreased fluorescence.

The fluorogenic compounds may in themselves be either non-fluorescent ormeta-fluorescent (i.e., fluorescent in a distinctly different way, e.g.,either by color or intensity, than the corresponding enzyme-modifiedproducts) and appropriate wavelengths of excitation and detection, maybe used to separate the fluorescence signal developed by the enzymemodification from any other fluorescence that may be present.

A number of enzyme substrates for enzymes of diverse origins, eithernaturally occurring or synthetic in origin, may be used. These mayinclude fluorogenic 4-methylumbelliferyl derivatives (hydrolyzable to4-methylumbelliferone); derivatives of 7-amido-4-methyl-coumarin;diacetylfluorescein derivatives; and fluorescamine.

Derivatives of 4-methylumbelliferyl that may be used as the enzymesubstrate may include:4-methylumbelliferyl-2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-lucopyranoside;4-methylumbelliferyl acetate;4-methylumbelliferyl-N-acetyl-beta-D-galactosaminide;4-methylumbelliferyl-N-acetyl-alpha-D-glucosaminide;4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide;2′-(4-methylumbelliferyl)-alpha-D-N-acetyl neuraminic acid;4-methylumbelliferyl-alpha-L-arabinofuranoside; 4-methylumbelliferylalpha-L-arabinoside; 4-methylumbelliferyl butyrate;4-methylumbelliferyl-beta-D-cellobioside;methylumbelliferyl-beta-D-N,N′-diacetyl chitobioside;4-methylumbelliferyl elaidate; 4-methylumbelliferyl-beta-D-fucoside;4-methylumbelliferyl-alpha-L-fucoside;4-methylumbelliferyl-beta-L-fucoside;4-methylumbelliferyl-alpha-D-galactoside;4-methylumbelliferyl-beta-D-galactoside;4-methylumbelliferyl-alpha-D-glucoside;4-methylumbelliferyl-beta-D-glucoside;4-methylumbelliferyl-beta-D-glucuronide; 4-methylumbelliferylp-guanidinobenzoate; 4-methylumbelliferyl heptanoate;4-methylumbelliferyl-alpha-D-mannopyranoside;4-methylumbelliferyl-beta-D-mannopyranoside; 4-methylumbelliferyloleate; 4-methylumbelliferyl palmitate; 4-methylumbelliferyl phosphate;4-methylumbelliferyl propionate; 4-methylumbelliferyl stearate;4-methylumbelliferyl sulfate; 4-methylumbelliferyl-beta-D-N, N′,N″-triacetylchitotriose; 4′-methylumbelliferyl2,3,5-tri-beta-benzoyl-alpha-L-arabinofuranoside;4-methylumbelliferyl-beta-trimethylammonium cinnamate chloride; and4-methylumbelliferyl-beta-D-xyloside.

Derivatives of 7-amido-4-methylcoumarin that may be used as the enzymesubstrate may include: L-alanine-7-amido-4-methylcoumarin;L-proline-7-amido-4-methylcoumarin; L-tyrosine-7-amido-4-methylcoumarin;L-leucine-7-amido-4-methylcoumarin;L-phenylalanine-7-amido-4-methylcoumarin; and7-glutaryl-phenylalanine-7-amido-4-methylcoumarin.

Peptide derivatives of 7-amido-4-methyl coumarin that may be used as theenzyme substrate may include: N-t-BOC-Ile-Glu-Gly-Arg7-amido-4-methylcoumarin; N-t-BOC-Leu-Ser-Thr-Arg7-amido-4-methylcoumarin; N-CBZ-Phe-Arg 7-amido-4-methylcoumarin;Pro-Phe-Arg 7-amido-4-methylcoumarin; N-t-BOC-Val-Pro-Arg7-amido-4-methylcoumarin; and N-glutaryl-Gly-Arg7-amido-4-methylcoumarin.

Derivatives of diacetylfluorescein that may be used as the enzymesubstrate may include fluorescein diacetate, fluoresceindi-(beta-D-galacto-pyranoside), and fluorescein dilaurate.

Where the enzyme whose activity is to be detected isalpha-D-glucosidase, chymotrypsin, or fatty acid esterase, e.g., fromGeobacillus stearothermophilus, a fluorogenic enzyme substrate that maybe used may be 4-methylumbelliferyl-alpha-D-glucoside,7-glutarylphenylalanine-7-amido-4-methyl coumarin, or4-methylumbelliferyl heptanoate, respectively. Where the enzyme whoseactivity is to be detected is alpha-L-arabinofuranosidase, e.g., derivedfrom Bacillus subtilis, a fluorogenic enzyme substrate that may be usedmay be 4-methylumbelliferyl-alpha-L-arabinofuranoside. Where the enzymewhose activity is to be detected is beta-D-glucosidase, e.g., derivedfrom Bacillus subtilis, a fluorogenic enzyme substrate that may be usedmay be 4-methylumbelliferyl-beta-D-glucoside.

An enzyme substrate that may be used may be a chromogenic compoundcapable of being enzymatically modified to give a derivative chromophor,or a product which reacts with another compound of a like or differentkind to give a derivative chromophor, which chromophor has a differentor more intense color. The chromogenic compounds may be non-colored orcolored in a distinctly different way, e.g., either by color orintensity, than the corresponding enzyme-modified products. Thesechanges may be discernible by eye or require the use of color detectinginstrumentation. Appropriate wavelengths of excitation and detection, inmanners well known to users of colorometric instrumentation, may be usedto separate the colored signal developed by the enzyme modification fromany other color that may be present.

Chromogenic compounds that may be used as enzyme substrates that may beused may include 5-bromo-4-chloro-3-indolyl derivatives; nitrophenylderivatives; indoxyl derivatives; and phenolphthalein derivatives.

Derivatives of 5-bromo-4-chloro-3-indolyl that may be used may include5-bromo-6-chloro-3-indolyl acetate, 5-bromo-4-chloro-3-indolyl acetate,5-bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside,5-bromo-4-chloro-3-indolyl-1,3-diacetate,5-bromo-4-chloro-3-indolyl-beta-D-fucopyranoside,5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside,5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid,5-bromo-4-chloro-3-indolyl phosphate, and 5-bromo-4-chloro-3-indolylsulfate.

Derivatives of nitrophenyl that may be used may include p-nitrophenoland o-nitrophenol derivatives. These include diethyl-p-nitrophenylphosphate; di-p-nitrophenyl phosphate;p-nitrophenyl-2-acetamido-2-deoxy-3-O-beta-galactopyranosyl-beta-glucopyranoside; p-nitrophenyl-2-acetamido-2-deoxy-beta-glucopyranoside;p-nitrophenyl acetate; p-nitrophenyl-N-acetyl-beta-D-glucosaminide;p-nitrophenyl-beta-D-N, N′-diacetylchitobioside;p-nitrophenyl-alpha-glucopyranoside; p-nitrophenyl-alpha-maltoside;p-nitrophenyl-beta-maltoside; p-nitrophenyl-alpha-mannopyranoside;p-nitrophenyl-beta-mannopyranoside; p-nitrophenyl myristate;p-nitrophenyl palmitate; p-nitrophenyl phosphate;bis(p-nitrophenyl)phosphate; tris(p-nitrophenyl)phosphate;p-nitrophenyl-beta-glucopyranoside; p-nitrophenyl-beta-glucuronide;alpha-p-nitrophenylglycerine; p-nitrophenyl-alpha-rhamnopyranoside;p-nitrophenyl stearate; p-nitrophenyl sulfate;p-nitrophenyl-2,3,4,6-tetra-O-acetyl-beta-glucosaminide; p-nitrophenylthymidine mono-phosphate;p-nitrophenyl-2,3,4-tri-O-acetyl-beta-glucuronic acid methyl ester; andp-nitrophenyl valerate.

Useful o-nitrophenols may include o-nitrophenyl acetate,o-nitrophenyl-beta-glucoside and o-nitrophenyl-beta-D-glucopyranoside.Other useful nitrophenyl derivatives may includenitrophenyl-beta-fucopyranoside; nitrophenyl-alpha-galactopyranoside;nitrophenyl-beta-galactopyranoside; nitrophenyl butyrate; nitrophenylcaprate; nitrophenyl caproate; nitrophenyl caprylate; nitrophenyllaurate; and nitrophenyl propionate.

Indoxyl derivatives that may be used may include indoxyl-acetate;indoxyl beta-D-glucoside; 3-indoxyl sulfate; and 3-indoxyl phosphate.

Phenolphthalein derivatives that may be used may include:phenolphthalein dibutyrate; phenolphthalein diphosphate; phenolphthaleindisulfate; phenolphthalein glucuronic acid; phenolphthaleinmono-beta-glucosiduronic acid; phenolphthalein mono-beta-glucuronicacid; and phenolphthalein mono-phosphate.

The above-described chromogenic enzyme substrates may react directlywith an appropriate enzyme to produce a chromophor.

Additional enzyme substrates containing 1-naphthyl, 2-naphthyl andNapthyl-AS-BI derivatives may be employed if the derivative enzymemodified product is further reacted with a chromogenic reagent, such asdiazotized dyes, e.g., 1-diazo-4-benzoylamino-2,5-diethoxybenzene,1-diazo-4-benzoylamino-2,5-diethoxybenzene,p-diazo-2,5-diethoxy-N-benzoyalanine, 4-chloro-2-methylbenzene diazoniumchloride, and o-aminoazotoluene diazonium salt, to produce a chromophor.

Derivatives of 1-napthyl that may be used may include1-naphthyl-N-acetyl-beta-D-glucosaminide.

Derivatives of 2-naphthyl that may be used may include2-naphthyl-phosphate; 2-naphthyl-butyrate; 2-naphthyl-caprylate;2-naphthyl-myristate; L-leucyl-2-naphthylamide; L-valyl-2-naphthylamide;L-cystyl-2-naphthylamide; N-benzoyl-DL-arginine-2-naphthylamide;N-glutaryl-phenylalanine 2-naphthyl-amine; 2-naphthyl-phosphate;6-Br-2-naphthyl-alpha-D-galacto-pyranoside;2-naphthyl-beta-D-galacto-pyranoside; 2-naphthyl-2-D-glucopyranoside;6-bromo-2-naphthol-beta-D-glucopyranoside;6-bromo-2-naphthyl-2-D-mannopyranoside; and2-naphthyl-alpha-L-fucopyranoside.

Derivatives of naphthyl-AS-BI that may be used may includenaphthyl-AS-BI-phosphate; and naphthyl-AS-BI-beta-D-glucuronide.

Where the enzyme whose activity is to be detected isalpha-D-glucosidase, e.g., from Geobacillus stearothermophilus, theenzyme substrate may be p-nitrophenyl-alpha-glucopyranoside. Where theenzyme whose activity is to be detected is alpha-L-arabinofuranosidase,e.g., derived from Bacillus subtilis, the enzyme substrate that may beused may be p-nitrophenyl-alpha-L-arabinofuranoside. Where the enzymewhose activity is to be detected is beta-D-glucosidase, e.g., derivedfrom Bacillus subtilis, the enzyme substrate that may be used may bep-nitrophenyl-beta-D-glucopyranoside. Where the enzyme whose activity isto be detected is β-galactosidase, the enzyme substrate may be5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. Where the enzyme whoseactivity is to be detected is β-galactosidase, the enzyme substrate maybe 4-methylumbelliferone-β-D-galactopyranoside.

The enzyme substrate that may be used may depend upon the identity ofthe enzyme whose activity is under study. Below is a list of a number ofenzyme substrates and corresponding enzymes which may react with thesubstrate to produce a product having appreciably modified or increasedfluorescence or color.

Enzyme Substrate Enzyme 4-Methylumbelliferyl acetate Esterase4-Methylumbelliferyl butyrate Esterase 4-Methylumbelliferyl elaidateLipase 4-Methylumbelliferyl-β-D- β-D-Galactosidase galactopyranoside4-Methylumbelliferyl-α-D- α-D-Galactosidase galactopyranoside4-Methylumbelliferyl-α-D- α-D-Glucosidase glucopyranoside4-Methylumbelliferyl-β-D- β-D-Glucosidase glucopyranoside4-Methylumbelliferyl heptanoate Esterase 4-Methylumbelliferyl oleateLipase 4-Methylumbelliferyl phosphate Acid or Alkaline Phosphatase4-Methylumbelliferyl propionate Esterase4-Methylumbelliferyl-β-D-galactoside β-D-Galactosidase4-Methylumbelliferyl-β-D-glucoside β-D-Glucosidase4-Methylumbelliferyl-α-D-glucoside α-D-Glucosidase4-Methylumbelliferyl-α-L- α-L-Arabinofuranosidase arabinofuranosideL-Leucine-7-amido-4-methylcoumarin Leucine aminopeptidase7-glutaryl-phenylalanine-7-amido-4- Chymotrypsin methylcoumarinD-Melibiose α-D-Galactosidase p-Nitrophenyl phosphate Alkaline or Acidphosphatase p-Nitrophenyl acetate Lipaseo-Nitrophenyl-β-D-galactopyranoside β-D-Galactosidasep-Nitrophenyl-α-D-galactopyranoside α-D-Galactosidaseo-Nitrophenyl-β-D-glucopyranoside β-D-Glucosidasep-Nitrophenyl-α-D-glucopyranoside α-D-Glucosidasep-Nitrophenyl-β-D-glucuronide β-D-Glucuronidasep-Nitrophenyl-α-L-arabinofuranoside α-L-Arabinofuranosidasep-Nitrophenyl laurate Esterase p-Nitrophenyl myristate Esterasep-Nitrophenyl palmitate Esterase p-Nitrophenyl phosphate diNa saltAlkaline Phosphatase Phenolphthalein dibutyrate Esterase Phenolphthaleindiphosphate Acid or Alkaline phosphatase Phenolphthalein diphosphatepentaNa salt Acid or Alkaline phosphatasePhenolphthalein-β-D-glucuronide Na salt β-D-GlucuronidasePhenolphthalein-β-D-glucuronide β-D-Glucuronidase L-Phenylalanineethylester HCl Chymotrypsin Phenyl-β-D-galactopyranosideβ-D-Galactosidase Phenyl-β-D-glucuronide β-D-GlucuronidasePhenyl-β-D-glucopyranoside β-D-Glucosidase Phenyl-β-D-glucuronideβ-D-Glucuronidase Phenyl-α-D-glucoside α-D-Glucosidase Sodiumβ-glycerophosphate Acid or Alkaline phosphatase Sodium 1-naphthylphosphate Acid or Alkaline phosphatase Sodium 2-naphthyl phosphate Acidor Alkaline phosphatase 2-Naphthyl-butyrate Esterase β-Naphthyl acetateLipase 6-Br-2-naphthyl-β-D-glucoside β-D-GlucosidaseL-Leucyl-2-naphthylamide aminopeptidase Leucine L-Valyl-2-naphthylamideaminopeptidase Valine N-glutaryl-phenylalanine-2-naphthylamineChymotrypsin Naphthyl-AS-BI-phosphate Phosphohydralase Indoxyl acetateLipase N-Methylindoxyl acetate Lipase N-Methylindoxyl myristate Lipase5-Bromoindoxyl acetate Lipase 3-Indoxyl phosphate Acid or Alkalinephosphatase Indoxyl-β-D-glucoside β-D-Glucosidase 5-Br-4-Cl-3-Indolylacetate Lipase 5-Br-4-Cl-3-Indolyl phosphate Alkaline or Acidphosphatase 5-Br-4-Cl-3-Indolyl-β-D-glucuronic acid β-D-GlucuronidaseDiacetylfluorescein Lipase/esterase

After the sterilization process has been completed, the carrier 12 withany enzyme that may have survived the sterilization process on it may becontacted with or placed in an aqueous medium or aqueous solution or ona semi-solid or solid containing an appropriate enzyme substrate. Theaqueous medium or aqueous solution may be buffered. An advantage of thedisclosed technology is that the amount of enzyme positioned on thecarrier 12 may be relatively small, and thus the volume of the enzymesubstrate needed to detect any enzymes that may have survivedsterilization may be relatively small. This may result in a detectionperiod that may be relatively short. The carrier 12 may have a surfacearea in the range from about 1 to about 80 mm², and in one embodiment inthe range from about 5 to about 50 mm². The units of enzyme supported bythe carrier 12, before sterilization, may be in the range from about10⁻⁷ to about 10⁶ units/mm², and in one embodiment in the range fromabout 10⁻⁴ to about 10³ units/mm². The enzyme and its appropriate enzymesubstrate may contact each other in the aqueous medium or aqueoussolution. An isotonic buffer, such as phosphate buffered salinesolution, tris(hydroxymethyl) aminomethane-HCl solution, or acetatebuffer may be used. These isotonic buffers may be compatible with mostfluorogenic and chromogenic enzyme substrates. Another consideration inchoosing the buffer is its influence on the enzyme activity. Forexample, phosphate buffered saline contains a high concentration ofinorganic phosphate which is a strong competitive inhibitor of alkalinephosphatase. For this enzyme, a tris-HCl buffer may be used.

The concentration of enzyme substrate in the buffered aqueous solutionmay be dependent upon the identity of the enzyme substrate and theenzyme, the amount of enzyme-modified product that must be generated tobe detectable, either visually or by instrument, and the amount of timerequired to determine whether active enzyme is present in the reactionmixture. The amount of enzyme substrate that may be sufficient may bethe amount needed to react with any active enzyme that may be presentafter the sterilization has been completed such that an enzyme-modifiedproduct at a molar concentration of at least about 1×10⁻⁸ molar may beproduced within a period of up to about 4 hours or less. Where theenzyme substrate is a 4-methylumbelliferyl derivative, its concentrationin the buffered aqueous solution may be in the range from about 1×10⁻⁵to about 1×10⁻³ molar.

The pH of the buffered aqueous solution containing the enzyme substratemay be adjusted to a pH in the range from about 5 to about 9.5, and inone embodiment about 7.5, in order to prevent autofluorescence for somebasic fluorogenic substrates.

The enzyme substrate in the aqueous buffered solution may be incubatedwith the enzyme whose activity is to be detected after the enzyme hasbeen subjected to the sterilization cycle. Incubation may be continuedfor a period of time and under conditions sufficient to liberate adetectable amount of the enzyme-modified product, assuming that any ofthe enzyme remains active. In general, the amount of product which maybe detectable may be as low as about 1×10⁻⁸ molar. The incubationconditions should be sufficient to generate at least about 1×10⁻⁸ molarof enzyme-modified product, and in one embodiment from about 1×10⁻⁶ toabout 1×10⁻⁵ molar of enzyme-modified product. The incubation time andtemperature needed to produce a detectable amount of enzyme-modifiedproduct may depend upon the identity of the enzyme and the enzymesubstrate, and the concentrations of each present in the bufferedaqueous solution. In general, the incubation time required may be up toabout 48 hours, and in one embodiment up to about 36 hours, and theincubation temperature may be in the range from about 20° C. to about70° C. The incubation time may be in the range from about 0.1 to about48 hours, and in one embodiment in the range from about 0.1 to about 36hours, and in one embodiment in the range from about 0.1 to about 24hours, and in one embodiment in the range from about 0.1 to about 12hours, and in one embodiment in the range from about 0.1 to about 6hours, and in one embodiment in the range from about 0.1 to about 4hours, and in one embodiment in the range from about 0.1 to about 3hours. Where Bacillus subtilis or Geobacillus stearothermophilus is thesource of the enzyme, the incubation time may be in the range from about0.1 to about 3 hours, and the incubation temperature may be in the rangefrom about 30° C. to about 40° C., and in the range from about 50° C. toabout 65° C., respectively.

Generally applicable methods for detecting the enzyme-modified productthat may be used may include photometric, potentiometric, gravimetric,calorimetric, conductometric, or amperometric techniques. In oneembodiment, fluorometric or spectrophotometric methods may be used. Forexample, the specific enzyme substrate may comprise a4-methylumbelliferyl derivative which on interaction with the enzymegives rise to umbelliferone which may be monitored fluorometrically, orthe substrate may comprise a nitrophenol, or similar type of derivative,which on interaction with the enzyme gives rise to a product which maybe monitored colorimetrically.

The biological indicator 30, although herein described primarily interms of a single enzyme and/or test organism, may comprise a pluralityof enzymes and/or test organisms or combinations thereof. For example,the biological indicator 30 may contain four types of enzymes (which maybe derived from three types of microorganisms), one enzyme beingresistant to heat, a second being resistant to gaseous sterilizingmedia, a third being resistant to radiation, e.g., gamma or betairradiation, and a fourth being resistant to fluid based media, e.g.,peracetic acid, stabilized hydrogen peroxide, chloramines, quaternaryamines, phenols, ozone water, or glutaraldehyde, etc. Similarly, thebiological indicator 30 may contain four species of test organisms, onespecies being resistant to heat, a second species being resistant togaseous sterilizing media, a third species being resistant to radiationand a fourth resistant to fluid based media.

Example 1

A sheet of Ahlstrom 238 paper (a paper product supplied by Ahlstrom) iscut into six circular support disks, each with a diameter of 6 mm(surface area of 28.3 mm²). Three of the disks are inoculated on oneside with 7 microliters of Geobacillus stearothermophilus sporesuspension having a population greater than 10⁸ cfu per milliliter.Three of the disks are not inoculated. Each disk is sonic welded to asupport strip of polystyrene having the dimensions of 25 mm×6 mm×0.25mm. For each disk, a weld horn is brought into contact with one side ofthe carrier strip while the disk is placed in contact with the otherside of the carrier strip. For the disks that are inoculated, the sideof the disks not containing spores is placed in contact with the supportstrip. Following sonication, the three uninoculated disks are inoculatedin the same manner as described above. FIG. 4 shows the results of thesetests. These tests demonstrate a lack of significant microorganism lossas a result of sonic welding.

Example 2

Sterilization indicator samples of the type described in Example 1 areinoculated with 10⁶ cfu/ml of Geobacillus stearothermophilus and exposedto incubation media samples of different volumes and bufferingcapacities. The samples are monitored for pH change as a function ofmicroorganism growth. The incubation media samples are as follows:

Sample A: 1.0 ml of Tryptic Soy Broth (TSB) with phenol red with fullbuffering capacity.

Sample B: 0.5 ml of TSB with phenol red and 0.5 ml of sterile deionized(DI) water with 50% of full buffering capacity.

Sample C: 0.4 ml of TSB with phenol red and 0.1 ml of sterile DI waterwith 80% of full buffering capacity.

Sample D: 0.3 ml of TSB with phenol red and 0.2 ml of sterile DI waterwith 60% full buffering capacity.

Sample E: 0.2 ml of TSB with phenol red and 0.3 ml of sterile DI waterwith 40% full buffering capacity.

The results are shown in FIG. 5. These results indicate that the smallerincubation media volumes and reduced buffering capacity show microbialgrowth more quickly than larger incubation media volumes.

While the disclosed technology has been explained in relation tospecific embodiments, it is to be understood that various modificationsthereof will become apparent to those skilled in the art upon readingthe specification. Therefore, it is to be understood that the inventiondisclosed herein is intended to cover such modifications as fall withinthe scope of the appended claims.

1. A process for making a sterilization indicator, the sterilizationindicator comprising: a carrier, the carrier having a first surface anda second surface, the first surface of the carrier having a surface areain the range from about 5 to about 50 mm²; a support, the support havinga first section and a second section, the carrier overlying the firstsection of the support, the second surface of the carrier being adheredto the first section of the support; and a biological indicatorsupported on the first surface of the carrier, the second section of thesupport being of sufficient dimension to permit handling thesterilization indicator without contacting the biological indicator,wherein the biological indicator comprises at least one test organism;the process comprising: applying the biological indicator to the carrierusing sonic welding, wherein the sonic welding, when applied to thecarrier with the biological indicator applied, exhibits no significantdifference in the biological indicator following the sonic welding ascompared to prior to the sonic welding; adhering the carrier to thesupport; and placing the support with the carrier adhered thereto into afirst compartment.
 2. The process of claim 1 further comprisingproviding a volume of incubation medium in a second compartment, thevolume of incubation medium being in the range from about 0.1 to about 5ml, the second compartment being adapted to maintain the incubationmedium separate from the sterilization indicator during sterilization,and the second compartment being adapted to permit the incubation mediumto contact the sterilization indicator after the sterilization indicatorhas been exposed to the sterilization medium, wherein the surface areaof the carrier and the volume of incubation medium enable a time ofincubation to determine whether a sterilization process was effective inthe range from about 0.1 to about 12 hours.
 3. The process of claim 2further comprising combining the first compartment and the secondcompartment in a sterilization kit.
 4. The process of claim 1 whereinthe carrier comprises paper, metal, glass, ceramic, plastic, membranes,compressed fibers or a combination of two or more thereof.
 5. Theprocess of claim 1 wherein the support is made of a material comprisingmetal, glass, ceramic, plastic, or a combination of two or more thereof.6. The process of claim 1 wherein the support is in the form of arectangular strip with a length, the first section of the supportcomprising a minor part of the length of the support, the second sectionof the support comprising a major part of the length of the support. 7.The process of claim 1 wherein the biological indicator furthercomprises one or more excipients.
 8. The process of claim 7 wherein theexcipient comprises one or more carbohydrates.
 9. The process of claim 7wherein the excipient comprises trehalose.
 10. The process of claim 7wherein the excipient comprises sucrose, glucose, maltose, dextran,starch, agarose, cellulose, protein, phosphonate, buffering agent, wax,lipid, oil, or a mixture of two or more thereof.
 11. The process ofclaim 1, wherein the concentration of organisms supported by the carrieris from about 10⁴ to about 10⁷ cfu per square millimeter of the carrier.12. The process of claim 1 wherein the carrier comprises paper, thebiological indicator comprises spores of Geobacillus stearothermophilus,and the support comprises polystyrene.
 13. The process of claim 1,wherein the biological indicator contains at least four species of testorganisms, comprising one species resistant to heat, one speciesresistant to gaseous sterilizing media, one species resistant toradiation and one species resistant to fluid sterilizing media.
 14. Theprocess of claim 1 wherein the biological indicator comprisesGeobacillus stearothermophilus.